Enterococcus faecalis is one of the top three most frequently-isolated species from hospital- associated infections of compromised patients and it can cause a highly fatal form of endocarditis. The medical importance of the enterococci is enhanced by their high degree of inherent and acquired resistance to antimicrobial agents, and by their propensity to act as a major conduit for the spread of resistance genes to more highly-pathogenic organisms. Enterococci are commonly isolated from biofilms formed on implanted medical devices, and colonization and growth on heart valves is also considered a form of biofilm growth. The biofilm environment is a likely niche for transfer of high-level vancomycin resistance from enterococci to Staphylococcus aureus. We have used genetic screens to identify a large number of novel genetic determinants of biofilm formation in vitro, and ongoing studies provide increasing evidence for the importance of many of these determinants in biofilms formed in vivo during infections. Many previously un-characterized enterococcal biofim determinants show significant conservation in a variety of important pathogens;if they are functionally conserved, the products of these genes comprise a pool of potential targets for the development of chemical inhibitors or vaccines that could block biofilm formation during infections produced by many different pathogens. The results from the current grant provide a strong foundation for answering important basic questions about the biology of enterococcal biofilm formation, as well as new approaches to increasing our understanding of bacterial pathogenic mechanisms. The specific aims proposed to address these questions are: 1) To use the genetic constructs and information from the present grant to identify critical signals and regulatory mechanisms involved in the earliest stages of biofilm development and in establishment and maintenance of the physiology of biofilm cells. 2) To test novel determinants of biofilm formation and virulence indentified in E, faecalis, and conserved in other gram-positive pathogens for functional conservation in biofilm formation and virulence in Staphylococcus aureus. 3) To screen the E. faecalis genome for determinants of biofilm-induced antibiotic resistance.